The Oral Ferroportin Inhibitor VIT-2763 Improves Erythropoiesis without Interfering with Iron Chelation Therapy in a Mouse Model of β-Thalassemia

In β-thalassemia, ineffective erythropoiesis leads to anemia and systemic iron overload. The management of iron overload by chelation therapy is a standard of care. However, iron chelation does not improve the ineffective erythropoiesis. We recently showed that the oral ferroportin inhibitor VIT-2763 ameliorates anemia and erythropoiesis in the Hbbth3/+ mouse model of β-thalassemia. In this study, we investigated whether concurrent use of the iron chelator deferasirox (DFX) and the ferroportin inhibitor VIT-2763 causes any pharmacodynamic interactions in the Hbbth3/+ mouse model of β-thalassemia. Mice were treated with VIT-2763 or DFX alone or with the combination of both drugs once daily for three weeks. VIT-2763 alone or in combination with DFX improved anemia and erythropoiesis. VIT-2763 alone decreased serum iron and transferrin saturation (TSAT) but was not able to reduce the liver iron concentration. While DFX alone had no effect on TSAT and erythropoiesis, it significantly reduced the liver iron concentration alone and in the presence of VIT-2763. Our results clearly show that VIT-2763 does not interfere with the iron chelation efficacy of DFX. Furthermore, VIT-2763 retains its beneficial effects on improving ineffective erythropoiesis when combined with DFX in the Hbbth3/+ mouse model. In conclusion, co-administration of the oral ferroportin inhibitor VIT-2763 and the iron chelator DFX is feasible and might offer an opportunity to improve both ineffective erythropoiesis and iron overload in β-thalassemia

The Oral Ferroportin Inhibitor VIT-2763 Improves Erythropoiesis without Interfering with Iron Chelation Therapy in a Mouse Model of β-Thalassemia

In β-thalassemia, ineffective erythropoiesis leads to anemia and systemic iron overload. The management of iron overload by chelation therapy is a standard of care. However, iron chelation does not improve the ineffective erythropoiesis. We recently showed that the oral ferroportin inhibitor VIT-2763 ameliorates anemia and erythropoiesis in the Hbbth3/+ mouse model of β-thalassemia. In this study, we investigated whether concurrent use of the iron chelator deferasirox (DFX) and the ferroportin inhibitor VIT-2763 causes any pharmacodynamic interactions in the Hbbth3/+ mouse model of β-thalassemia. Mice were treated with VIT-2763 or DFX alone or with the combination of both drugs once daily for three weeks. VIT-2763 alone or in combination with DFX improved anemia and erythropoiesis. VIT-2763 alone decreased serum iron and transferrin saturation (TSAT) but was not able to reduce the liver iron concentration. While DFX alone had no effect on TSAT and erythropoiesis, it significantly reduced the liver iron concentration alone and in the presence of VIT-2763. Our results clearly show that VIT-2763 does not interfere with the iron chelation efficacy of DFX. Furthermore, VIT-2763 retains its beneficial effects on improving ineffective erythropoiesis when combined with DFX in the Hbbth3/+ mouse model. In conclusion, co-administration of the oral ferroportin inhibitor VIT-2763 and the iron chelator DFX is feasible and might offer an opportunity to improve both ineffective erythropoiesis and iron overload in β-thalassemia

Presence of heat shock protein 70 in secondary lymphoid tissue correlates with stroke prognosis

Heat shock protein 70 (Hsp-70) can act as a danger signal and activate immune responses. We studied the presence of Hsp-70 in lymphoid tissue and plasma of acute stroke patients and asymptomatic controls free of neurological disease. Immunofluorescence, Western blotting, qRT-PCR and flow cytometry studies were performed. Plasma Hsp-70 concentration at day 7 was similar in patients and controls, whereas patients disclosed stronger immunoreactivity to Hsp-70 in lymphoid tissue than controls. Most Hsp-70. + cells were antigen presenting cells located in T cell zones. Stronger immunoreactivity to Hsp-70 was associated with smaller infarctions and better functional outcome. © 2014 Elsevier B.V.This work was supported by grants from the ‘Instituto de Salud Carlos III’ (ISCIII) (PI09/1313), the Spanish Ministry of Science and Innovation (SAF2011-30492), the ERANET-NEURON project (PRI-PIMNEU-2011-1342) of the European Community, and a donation by the Doctor Melchor Colet Foundation. We are indebted to IDIBAPS Biobank, Xarxa de Bancs de Tumors de Catalunya (XBTC) financed by ‘Pla Director d’Oncologia de Catalunya’ and Red Nacional de Biobancos (RNBB, ReTBioH) financed by ISCIII (RETIC RD09-0076/0038)Peer Reviewe

The prion protein is an agonistic ligand of the G protein-coupled receptor Adgrg6

Ablation of the cellular prion protein PrPC leads to a chronic demyelinating polyneuropathy affecting Schwann cells. Neuron-restricted expression of PrPC prevents the disease, suggesting that PrPC acts in trans through an unidentified Schwann cell receptor. Here we show that the cAMP concentration in sciatic nerves from PrPC -deficient mice is reduced, suggesting that PrPC acts via a G protein-coupled receptor (GPCR). The amino-terminal flexible tail (residues 23-120) of PrPC triggered a concentration-dependent increase in cAMP in primary Schwann cells, in the Schwann cell line SW10, and in HEK293T cells overexpressing the GPCR Adgrg6 (also known as Gpr126). By contrast, naive HEK293T cells and HEK293T cells expressing several other GPCRs did not react to the flexible tail, and ablation of Gpr126 from SW10 cells abolished the flexible tail-induced cAMP response. The flexible tail contains a polycationic cluster (KKRPKPG) similar to the GPRGKPG motif of the Gpr126 agonist type-IV collagen. A KKRPKPG-containing PrPC -derived peptide (FT 23-50) sufficed to induce a Gpr126-dependent cAMP response in cells and mice, and improved myelination in hypomorphic gpr126 mutant zebrafish (Danio rerio). Substitution of the cationic residues with alanines abolished the biological activity of both FT 23-50 and the equivalent type-IV collagen peptide. We conclude that PrPC promotes myelin homeostasis through flexible tail-mediated Gpr126 agonism. As well as clarifying the physiological role of PrPC, these observations are relevant to the pathogenesis of demyelinating polyneuropathies - common debilitating diseases for which there are limited therapeutic options

Ionomics

<p>Trace elements in their ionic form mediate biochemical reactions in human cells by acting as enzyme cofactors or centers for stabilizing protein structures. Deficit or accumulation of these substances lead to cell toxicity and severe diseases in humans and therefore, intracellular trace ion concentrations (i.e. the "ionome") must be tightly controlled. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine and quantify the intracellular trace ion concentrations. In ionomics assays HEK cells overexpressing a particular doxycycline-inducible SLC transporter were used to quantify the change of inorganic ions by ICP-MS upon cell lysis. ICP-MS based ionomics is rather a low throughput assay, as a significant volume of sample is required.</p&gt

The oral ferroportin inhibitor vamifeport improved hemodynamics in a mouse model of sickle cell disease

Sickle cell disease (SCD) is an inherited hemolytic anemia caused by a single point mutation in the beta‑globin gene of hemoglobin that leads to synthesis of sickle hemoglobin (HbS) in red blood cells (RBCs). HbS polymerizes in hypoxic conditions, leading to intravascular hemolysis, release of free hemoglobin and heme, and increased adhesion of blood cells to endothelial vasculature, which causes painful vaso-occlusion and organ damage. HbS polymerization kinetics are strongly dependent on the intracellular HbS concentration; a relatively small reduction in cellular HbS concentration may prevent HbS polymerization and its sequelae. We hypothesized that iron restriction via blocking ferroportin, the unique iron transporter in mammals, might reduce HbS concentration in RBCs, thereby decreasing hemolysis, improving blood flow, and preventing vaso-occlusive events. Indeed, vamifeport (also known as VIT-2763), a clinical-stage oral ferroportin inhibitor, reduced hemolysis markers in the Townes model of SCD. The RBC indices of vamifeport-treated male and female Townes (HbSS) mice showed changes attributable to iron-restricted erythropoiesis: decreased corpuscular hemoglobin concentration mean and mean corpuscular volume, as well as increased hypochromic and microcytic RBC fractions. Furthermore, vamifeport reduced plasma soluble vascular cell adhesion molecule-1 concentrations, which suggests lowered vascular inflammation. Accordingly, intravital video microscopy of fluorescently labeled blood cells in the microvasculature of Townes mice treated with vamifeport demonstrated diminished adhesion to the endothelium and improved hemodynamics. These preclinical data provide a strong proof-of-concept for vamifeport in the Townes model of SCD and support further development of this compound as a potential novel therapy in SCD

One route of SC migration: the blood vessels.

<p>SC grafted in the spinal cord parenchyma are often localized in white matter around blood vessels (asterisks), evidenced with anti-laminin antibody (blue). While at 7 days SC are present close to the blood vessel wall (A), at 21 days they are embedded in the perivascular space but remote from the vascular wall (B).</p

Distribution of GFP labeled SC after delivery in the cisterna magna and the spinal cord.

<p>GFP-SC (green) are detected both in the cerebellar parenchyma (A, B) and meninges (C) 7 days (A–C) after cisterna magna delivery as well as 21 days after in the proximal spinal cord (D). GFP-SCs grafted in the spinal cord parenchyma (E, F) are concentrated around blood vessels, some migrate away from the graft toward a lesion (L) identified by MOG immunostaining through white matter (E, arrows). (F) Same field illustrating GFP-SCs, inset is a higher magnification.</p

<i>In vitro</i> characterization of transduced SC-GFP.

<p>The majority of SC-GFP (green, A, C) express the SC marker p75 (red, B, C).</p

Interaction of GFP-SCs with myelin and astrocytes after delivery in the cisterna magna or the spinal cord parenchyma.

<p>Combined detection of MOG, GFAP and GFP on cryostat sections showed that GFP-SCs grafted in the cisterna magna (A, D) or the spinal cord (B,C, E, F) are found in demyelinated lesions (delineated by dashed lines) detected by MOG immunostaining (red) both after 7 days (B) and 21 days (A, C) after delivery. GFAP+ astrocytes (blue) interact with GFP-labeled SCs in the cisterna magna (D) and the spinal cord (E, F); 7 days (E) or 21 days (F) after spinal cord graft or 21 days after cisterna magna graft (D).</p